MetaTopolin-driven breakthrough in lemon beebalm (Monarda citriodora) regeneration: A molecular fidelity study for genome engineering applications

Plant cell and tissue regeneration has become the key platform for genome editing and integrated approaches. However, there is a lack of reliable and reproducible in-vitro conservation methods for several plant species. In addition, collecting plant material from natural habitats has decreased species numbers, prompting efforts to create growing methods that support conservation. Biotechnological approaches such as plant tissue culture have increasingly satisfied this growing demand. Thus, optimization of in-vitro regeneration system in Monarda citriodora Cerv. ex-Lag., was done using nodes as explants. The nodal explants were cultured on Murashige and Skoog’s (MS) medium blended with different concentrations of 6-benzylaminopurine (BAP), kinetin (Kn), Thidiazuron (TDZ), metaTopolin (mT) alone or in combination. The highest regeneration efficiency (90.31 ± 0.15%) with multiple shoots (13.22 ± 0.26) induction and shoot length (4.32 ± 0.06 cm) was achieved on shoot regeneration medium (SRM) containing MS salts fortified with 1 mg/L mT. Furthermore, we found that the combined application of 1 mg/L mT+ 0.5 mg/L α-Naphthaleneacetic acid (NAA) resulted in an increased shoot number (25.30 ± 0.45) with a shoot length of 6.13 ± 0.02 cm. A higher rooting percentage (85.51 ± 0.42) was recorded in plants grown in half-strength MS media. The start codon targeted (SCoT) and inter simple sequence repeat (ISSR) markers revealed that the in-vitro regenerated and greenhouse-acclimatized plants showed a similar banding pattern to that of the in-vivo grown mother plant, thus confirming the absence of a polymorphic nature of regenerated M. citriodora plants. These results signify that mT enhances the regeneration and development rate of M. citriodora plants. Keywords: Molecular markers, Monarda citriodora, MS basal, Nodal explants, Regeneration